ABSTRACT: Although carbon isotopes in fish otoliths are widely utilized to obtain information on environmental records, uncertainty regarding the sources of otolith carbonate, either from fish diet or ambient water, limits detailed determinations. The present study experimentally determined absolute incorporation rates of carbon derived from 2 sources, metabolic CO2 and ambient water, into otoliths using goldfish under controlled conditions. In addition, the proportions of the 2 sources in otolith daily increments were also determined by the diel variations in the rate of incorporation from the 2 sources. A group of fish was administered with d-[14C-U]-glucose or was exposed to water containing NaH14CO3, and incubated for 1, 3, 6, 12 and 24 h. Another group was similarly treated with the radiocarbons at 06:00, 12:00, 18:00, 00:00 or again at 06:00 h, and were incubated for 6 h. After incubation, serum and otoliths were collected and were separated into organic and inorganic carbon fractions, and the incorporation rates from the 2 sources were determined. The rates of carbon incorporation from d-[14C-U]-glucose injected and NaH14CO3 water were 0.27 (25.5%) and 0.79 (74.5%) nmol mg1 otolith h1, respectively. During darkness (18:00 to 06:00 h), both the incorporation rate and proportion of metabolic-CO2-derived carbon significantly decreased to ~50% of that during daylight. These results suggest that otolith carbonate is mainly derived from ambient water, and the ratio of carbon derived from metabolic CO2 is lower in the D-zone than that in the L-zone of the otolith daily increments.